Several million units of donor platelets are transfused annually in the US and Europe to treat patients undergoing radiation treatment, chemotherapy, or organ transplant surgery. Although transfusions of donor- derived platelets are an effective way to treat thrombocytopenic patients, numerous concerns have been raised due to the increase in demand, the five-day shelf-life, and the possibilities for contamination. Platelets are produced by megakaryocytes (MKs) in the bone marrow and are derived from hematopoietic stem and progenitor cells (HSPCs). The long-term objective of this proposal is to generate platelets ex vivo from HSPC- enriched umbilical cord blood that will allow us to address a vast public need. Recent studies have shown that the addition of histone deacetylase inhibitors (HDACi) can greatly affect expansion of progenitor cells, but can reduce MK differentiation efficiency. My central hypothesis is that epigenetic and transcriptional changes prime or desensitize HSPC to MK differentiation cues. To investigate the role of epigenetic signals during HSPC-to- MK transition, I propose to perform high-throughput ChIP and RNA-sequencing to discover differentially active regulatory elements between expanded (low potential) and fresh cord blood HSPCs. This highly interdisciplinary and collaborative proposal utilizes computational biology and machine-learning to identify differentially active regulatory elements, and associate genetic features with MK fate. To validate my findings and establish a causal relationship between histone modifications of certain regions and MK potential, I will generate a dCas9-fusion protein that can directly manipulate the epigenetic marker at a targeted site. My findings will establish the mechanism of action of chromatin-modifying agents such as HDACi, and the causal relationship between the epigenome and MK differentiation. My highly collaborative proposal with the Miller, Bagheri, and Mahmud lab will substantially enhance our understanding of hematopoietic differentiation toward manufacturing platelets.